University of Sydney Library Electronic Item MBLG2x71 Author of lecture: Professor A.S. Weiss Title of lecture: L2.

Transcriptional Regulation I

MBLG2x71
9 March 2012

Lecture 2 Transcriptional Regulation I

Professor Tony Weiss
Models of Gene Expression: the lac operon. Uses of the lac operon in Molecular Biology, blue/white colour selection Pages 553-554, 559-561 Watson et al. Molecular Biology of the Gene 6th Edn.

With thanks to: R. Benjamin, UNT USA

Gene expression responds to environmental conditions

Some regulatory proteins present at 5 510 copies per cell, some enzymes for glycolysis present at 100,000 copies/cell Genes whose products are presently unneeded or at acceptable levels are turned off Mechanisms to achieve proper mix of gene expression are varied

Lactose metabolism in E. coli

Jacob and Monod (1946) studied as model system, many others followed Enzyme -galactosidase only expressed when lactose present in the medium Enzyme said to be expressed in inducible fashion with lactose as inducer Identified cis-acting elements (operator, promoter) and trans-acting factors

Lactose Hydrolysis
Enzyme encoded by lacZ gene Glucose and galactose products y Enzyme cleaves broad range of galactosides
Including synthetic analogs such as X-gal

Bacterial Genes and Operons

Related genes are often clustered and expressed as a unit on a single mRNA
Operon Polycistronic mRNA

E. coli lac operon

lacI has a constitutive promoter and is expressed separately from the lac operon
Encodes lac repressor Low level of expression

lac operon expression

lac operon encodes polycistronic mRNA giving rise to 3 different enzymes

Structural Genes of lac Operon

Structural genes encode the primary structure of the enzymes/proteins
For lac operon these are lacZ, lacY and lacA Enzymes encoded are -galactosidase, lactose permease and transacetylase, respectively Genes are also coordinately regulated

lac Operon Expression

lac Operon Expression

In absence of inducer, repressor binds to operator and blocks RNAP from binding to promoter

lac Operon Expression

Binding of inducer to repressor causes conformational change in protein, preventing interaction with operator RNA polymerase binds to promoter and expresses operon

Gratuitous Inducers
Lactose is normal inducer (actually allolactose for the pure at heart)
But other -galactosides also work

Isopropylthiogalactoside (IPTG) also acts as an inducer but is not metabolized

Shows induction does not involve interaction with the actual enzyme being synthesized

Isopropylthiogalactoside (IPTG)
Gratuitous inducer Not metabolised Level remains co s a constant

The lac promoter is quite weak

Problem: Thats OK if some glucose is around but: That s
if lactose if available (and glucose isnt available) cells could get very hungry!

Solution: when cells are hungry:

make the lac promoter work more effectively

Mechanism: CAP (catabolite activator protein) also called CRP (cAMP receptor protein)

10

(CAP and CRP are the same)

Not enough glucose

CAP binds cAMP then binds the promoter

11

With glucose

CAP needs cAMP to enable it to bind promoter (conformation is incorrect for binding)

12

Xgal is incredibly useful!

and theres a movie

www.youtube.com/watch?v=oBwtxdI1zvk

13